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Molecular Biology Flashcards

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1
Q

Typical make up of a human cell

A

50% protein
40% lipids
10% carbohydrates

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2
Q

Building blocks of a cell

A

Carbon chains
Sugars
Amino acids
Sugars and bases

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3
Q

Macromolecules of a cell

A

Lipids
Complex carbohydrates
Proteins
Nucleic acids RNA/DNA

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4
Q

What do macromolecules form

A

Supramolecular assemblies (membranes, ribosomes, chromosomes) and organelles

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5
Q

What is the process of molecule production

A

Step by step process with different enzymes catalysing each step to create final products

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6
Q

How is the process of making cellular molecules regulated

A 
Transcription control
RNA processing and stability
Translational control
Protein processing
Protein activity and stability
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7
Q

What is transcriptional control

A

Way which controls when and in what cells genes are transcribed and produce mRNA and then proteins

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8
Q

Breakdown of DNA and genes in different cells

A

Each cell has same DNA and ~21,000 coding genes
10,000 genes are expressed in all cells
~1000-2000 unique coding genes for specific cell types
Rest expressed in some but not all cells

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9
Q

How do cells have unique functions and structures

A

Each cell contains specific genes which are expressed so cause different proteins and different functions

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10
Q

What are transcription factors

A

Proteins that bind to a specific DNA sequence and control the rate of transcription. Bind tightly to DNA when a particular sequence is recognised, interact with one another and RNA polymerase to determine if a gene is turned on or off

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11
Q

What is the promotor region/ regulatory region

A

DNA sequence which transcription factors bind to and recruit RNA polymerase and determines if and how much gene is transcribed

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12
Q

What is the transcribed region of a gene

A

Sequences of DNA that are transcribed into pre-mRNA which is then processed so it can be translated by having its intron sequences removed

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13
Q

How do transcription factors regulate transcription

A

Gene is only transcribed if both activator transcription factors are present and repressor is absent. They interact with DNA, one another and RNA polymerase. Ensures transcription only occurs under the right conditions

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14
Q

How doe pancreatic B cells and liver cells differ to perform their different functions

A

Pancreatic B cells only have one type of transcription factor which works to turn on insulin secretion gene and liver cells contain another type of transcription factor which works to turn on glucagon synthesis gene

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15
Q

How can hormones and other extracellular material affect transcription with transcription factors

A

When transcription needs to stop or not occur, these extracellular materials can bind to receptors and cause gene expression to not occur by changing/ affecting the proteins inside the cell or cause repressor to bind to DNA

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16
Q

mendels laws of inheritance

A

Law of segregation
Law of independent assortment and recombination
Law of dominance

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17
Q

What is the law of segregation

A

When gametes form, alleles are separated so that each gamete carries only one allele for each gene

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18
Q

What is the law of independent assortment

A

Segregation and recombination of alleles for one gene occurs independently to that of any other gene

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19
Q

What is the exception to the law of independent assortment

A

Linked genes/ genetic linkage

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20
Q

What is the law of dominance

A

Some alleles are dominant and some are recessive. Organisms with atleast one dominant allele show the dominant trait

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21
Q

Why are we different in respect to genetic differences?

A

We inherit a unique combination of alleles from our parents. DNA differs at ~20 million places and 5% of these differences (1 million) might affect how a gene functions

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22
Q

How can a genetic disorder from a dysfunctional protein alter a pathway causing a disease

A

Proteins in the pathway are not made so therefore reactions cant proceed and build ups of products occur which cant be used to form final products or more products in the pathway

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23
Q

How do we differ in respect to environment

A

Environment determines how some genetic differences affect us

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24
Q

Example of how genetics and environment can cause the same genetic disorder to affect people differently

A

Phenylalanine build-up from a loss of protein in the reaction pathway

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25
How does the environment affect phenylalanine build up differently between people
Diet. Low phenylalanine diet prevents intellectual disability and associated problems in a PRU individual
26
How does genetics affect phenylalanine build up differently between people
Phenylalanine is transported across blood to brain barrier. Genetic variation means efficiency of transporter differs between individuals so individuals with less efficient transporters have lower phenylalanine levels in the brain meaning less damage
27
Does a mutation in a gene at the start or towards the end cause more harmful outcomes and why
Towards the start because more of the products along the pathway can be used for different things, not just as a part of that pathway so can affect the body in many ways
28
What are germline mutations
Occur in gametic cells and can be passed on to future generations
29
What are somatic mutations
Occur in body cells, not passed on to future generations and can result in cancer
30
How many cells make up the human body and when do they replicate
~37 trillion. | Die when cells are old or damaged and are replaced by new replicated ones
31
What is cancer
Multiple mutations and a collection of diseases which cause cells to no longer respond to signals that control cell growth and death. Causes more rapid growth and replication. In later stages they break through normal tissue and metastasise to new sites in the body
32
Why does the risk of cancer increase with age. What prevents this
The more times a cell divides, the more likely it is to gain a mutation. Normal cell replications are limited to control division and try prevent this
33
What can cause a somatic cell mutation
Spontaneous by chance or exposure to a mutagen (known as carcinogen)
34
What can cause cancer
Mutations/ bad luck/ chance and environment factors and lifestyle can contribute and increase the chance eg smokers are 23 times more likely to get cancer
35
What two types of genes can cause cancer when mutated
Tumor suppressor genes and onco-genes
36
How can a mutation in tumor suppressor genes cause cancer
Encode proteins (such as inhibitory proteins and proteins that prevent other mutations) which prevent uncontrolled growth get mutated and results in a recessive loss of function mutation where they no longer work.
37
How can a mutation in onco-genes cause cancer
Encode proteins (such as proteins that stimulate cell division) become mutated resulting in a dominant gain of function mutation which promotes cell division and promotes cancer
38
How does chronic mild leukemia occur
Oncogene dominant mutation causes constant activation of BCR-ABL protein so constant transcription factor. Suppressor recessive mutation either causing constant active E2F transcription factor or constant kinase phosphorylation from p16 mutation
39
Likelihood of having the suppressor mutation if have the oncogene mutation
~1/2 of people have E2F mutation and ~20% have p16 mutation
40
What is the genetic predisposition
Inherited mutations increase the chance of someone getting cancer as they already have a mutation, easier to get the others
41
What are the different cancer treatments
Surgery to remove cancer cells Radiation therapy which targets and kills cancer cells Chemotherapy- drugs that target cancer cells, also affect normal body cells Targeted therapy- drugs that target changes in cancer cells that allow them to grow and divide/ target cancer specific mutations
42
An example of a targeted therapy
Gleevac inhibits the kinase activity of BCR-ABL so it doesn’t constantly activate the transcription factor
43
Why does cattle and pig insulin not work well in human bodies and what happens
Amino acid sequence isnt identical so isn’t always 100% pure in humans. Causes range of immune responses from local irritation to anaphylactic shock
44
What do recombinant technologies do and what is most commonly used
Joining bits of DNA together which is then inserted into an organisms to produce and express a useful protein. Plasmids most commonly used
45
What is a plasmid
Usually circular pieces of DNA which aren’t a part of chromosomal DNA and replicate independently to chromosomal DNA. Common in bacteria but can be found in eukaryotes and can provide benefits to hosts such as antibiotic resistance
46
What are key components of recombinant DNA plasmids and what does each component do
Origin of replication (ORI) allows initiation of replication with DNA polymerase Antibiotic resistance gene allows for antibiotic selection Promotors drives expression of selected and favoured genes in cells with appropriate transcription factor machinery. Can be generic of specific to specific cell types Also contains 2 restriction sites
47
3 tools used for recombinant DNA technologies
Restriction enzymes, DNA ligases and transformation
48
How do restriction enzymes and DNA ligases work/ what do they do
Cut and pasting of DNA into plasmids. Restriction enzymes are found in bacteria (to degrade foreign DNA) and cut specific regions of DNA. DNA ligase attaches sticky ends of plasmid and introduced DNA with complementary base pairing and catalyses formation of the phosphodiester bond to repair the backbone
49
How does transformation work
Transfers plasmids into bacteria and transformed bacteria are then selected by antibiotic resistance/ antibiotic selection. Amplification and reproduction of the selected for bacteria with antibiotic resistance occurs. DNA is purified for downstream uses such as PCR, cloning, transfection into other organisms
50
What does a universal genetic code mean and what is its significance
All organisms read the same codons as the same amino acids. Means that we can transform human genes into bacteria and it will still make the same protein
51
What needs to be considered when cloning eukaryotic genes in prokaryotes. Why is the same thing done when transferring eukaryotic genes to eukaryotes aswell?
Prokaryotes dont contain introns so dont have the mechanisms to remove them from DNA when replicating. This means that introduced DAN must be the coding mRNA without the introns. Done for eukaryotes too as when introns are included the plasmids can be very long and too hard to work with
52
What are the five steps in producing a recombinant protein
Isolate gene of interest Clone into expression plasmid Transform into bacteria for expression or isolation of more DNA for use in another expression system Grow cells expressing protein of interest eg antibiotic selection Isolate and purify protein
53
How is recombinant insulin made in a prokaryotic cell
Cut and paste to isolate gene of interest A and B parts are separated and put into different plasmids as insulin is a pre-pro-protein which requires further processing in golgi and this cant happen in prokaryotes Plasmids are placed into two different bacterial cells Bacteria undergo antibiotic selection A and B extracted by purifying insulin fusion proteins, treated with cyanogen bromide to cleave A and B, then purify and mix A and B and form disulfide bonds to get functional insulin
54
Advantages and disadvantages of using prokaryotic systems for recombinant proteins
Advantages: relatively low cost, high yield and pathogen free Disadvantages: proteins are often partially folded and inability to perform post-translational modifications
55
Why would we make recombinant proteins such as insulin in mammalian cells?
Protein can be produced as pre-pro-protein and processed efficiently. It will also be secreted from cells meaning easier purification
56
Downside of producing proteins such as insulin in mammalian cells
More expensive to produce
57
How will insulin production in mammalian/ eukaryotic cells be different to that of prokaryotic cells
Isolation of cDNA done the same way, cloned into eukaryotic expression plasmid, transformed into bacteria to produce more plasmids and then transfected into eukaryotic cells, recombinant insulin is extracted from media and then purified
58
What are therapeutic proteins
Recombinant human proteins which are only active when post-translational modification occurs via glycosylation which requires mammalian cells
59
Example of a therapeutic protein and what it does and is used for
Erythropoietin (EPO). Increases RNC count so increases oxygenation of muscles. Used for doping and to treat disease states that reduce RBC count such as chronic renal failure and chemotherapy which can both lead to anaemia
60
When was EPO first cloned and how is it done
First cloned in early 1980s | Made in Chinese hamster ovary (CHO)
61
What is pharming and why would we use it
Transgenesis/ using whole animals to make recombinant proteins. Used because cells in culture can’t perform all post-translational modifications
62
Example of pharming
Y-carboxylation of certain glutamate residues. First recombinant protein transgenic animal was approved as a drug was anti-thrombin (AT) which is expressed in milk of a transgenic goat at lactation and then purified from other milk products
63
What results from AT deficiency, what is its frequency and how can it occur
Leads to increased risk of inappropriate blood clotting. Frequency is 1/2000-5000 and may be hereditary or acquired
64
how are new sets of enzymes generated
Take enzymes and make random mutations, clone mutated enzymes into plasmid of choice, use promotor in bacterial or mammalian cells depending on if post-translational modification is required
65
What is gene therapy and why is it beneficial in humans
Where humans are used as the host for protein production. Plasmids can be carried into patients using viral vectors which have been deleted of their own genome. Promotor in the plasmid is specific to the cell type so that it can get to target organ unharmed. Causes expression for life as protein becomes part of the DNA due to viral activity injecting its DNA= once-off treatment
66
How can gene therapy be used for diabetes and what has it successfully been tested on
Promotor is glucose responsive and liver specific | Pre-proinsulin cDNA used- not mRNA with introns as is would be too big for viral vector
67
What can be used to edit humans and the bad effects of this | SLIDE SAID NOT EXAMINABLE BUT OH WELL
CRISPR-Cas9; breaks DNA and fixing by proteins causes mutations from no template DNA, if template provided then can choose DNA sequence and phenotype. Useful in fixing disease and is based on bacterial immune system Detrimental to human evolution and possibly natural selection

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